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A chloroplast thylakoid lumen protein is required for proper photosynthetic acclimation of plants under fluctuating light environments Jun Liua and Robert L. Lasta,b,1 a Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824; and bDepartment of Plant Biology, Michigan State University, East Lansing, MI 48824

Despite our increasingly sophisticated understanding of mechanisms ensuring efficient photosynthesis under laboratory-controlled light conditions, less is known about the regulation of photosynthesis under fluctuating light. This is important because—in nature— photosynthetic organisms experience rapid and extreme changes in sunlight, potentially causing deleterious effects on photosynthetic efficiency and productivity. Here we report that the chloroplast thylakoid lumenal protein MAINTENANCE OF PHOTOSYSTEM II UNDER HIGH LIGHT 2 (MPH2; encoded by At4g02530) is required for growth acclimation of Arabidopsis thaliana plants under controlled photoinhibitory light and fluctuating light environments. Evidence is presented that mph2 mutant light stress susceptibility results from a defect in photosystem II (PSII) repair, and our results are consistent with the hypothesis that MPH2 is involved in disassembling monomeric complexes during regeneration of dimeric functional PSII supercomplexes. Moreover, mph2—and previously characterized PSII repair-defective mutants—exhibited reduced growth under fluctuating light conditions, while PSII photoprotection-impaired mutants did not. These findings suggest that repair is not only required for PSII maintenance under static high-irradiance light conditions but is also a regulatory mechanism facilitating photosynthetic adaptation under fluctuating light environments. This work has implications for improvement of agricultural plant productivity through engineering PSII repair.

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fluctuating light photosynthesis photosystem II repair photoprotection chloroplast thylakoid lumen

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nonphotochemical quenching (NPQ) (9, 10) and also control the rate of photosynthetic electron flow through cytochrome b6f to safeguard PSI under fluctuating light conditions (11). Despite operation of these protective mechanisms, the photosynthetic apparatus—and especially PSII—is prone to damage at all light intensities. Plants also have a robust repair system that replaces damaged PSII reaction center proteins (12, 13), and a set of proteins facilitate this longer term repair process (14–16). Other long-term light responses include regulating expression of photosynthesisrelated genes (17–20) and modulating the abundance of key photosynthetic proteins as well as the stoichiometry of photosystems to optimize light energy capture and conversion (21, 22). Despite the increasing sophistication of our understanding of how plants avoid, minimize, or repair photodamage, the majority of regulatory mechanisms have been characterized under controlled environmental conditions. Little is known about how photosynthetic energy conversion is regulated to ensure optimal plant performance in natural dynamic environments. We describe characterization of an Arabidopsis thaliana chloroplast thylakoid lumen protein, MPH2 (MAINTENANCE OF PHOTOSYSTEM II UNDER HIGH LIGHT 2, encoded by At4g02530), which is required for photosynthetic acclimation to greenhouse fluctuating light environments as well as to short- and long-term constant high light. Biochemical and molecular analyses provide evidence suggesting that MPH2 is a disassembly factor Significance

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lants and photosynthetic microbes harness sunlight for photosynthesis to supply oxygen, food, fuel, and fiber. However, sunlight quality and quantity vary across the globe and change in timescales from seconds to seasons. Plants are dependent on sunlight for photosynthesis yet must cope with dynamic light fluctuations. In fact, deleterious effects on photosynthesis and plant performance can result from mismatch between the amount of incident sunlight and the capacity of the photosynthetic machinery to use that energy. Plants have evolved physiological mechanisms enabling the photosynthetic apparatus to adapt to natural, everchanging light environments (1). Exploring regulatory mechanisms that ensure high photosynthetic efficiency and productivity under fluctuating light can contribute to sustainable agriculture at a time of changing climate and continuous population growth (2, 3). Plants use diverse strategies to regulate the efficiency of photosynthesis in response to changes in light conditions. These responses can be classified into two general categories based on the timescale of action: short-term (seconds to minutes) and longterm (minutes to hours or longer). In the short term, plants adjust the distribution of excitation energy between photosystems I and II (PSI and PSII), a regulatory mechanism known as state transition (4, 5). This process is regulated by reversible phosphorylation of the mobile fraction of major light-harvesting complex II (LHCII) via STN7 kinase and PPH1/TAP38 phosphatase (6, 7). Photoprotective mechanisms are another important short-term strategy (8). Plants dissipate excess light energy as heat through

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Photosynthesis harnesses sunlight to assimilate carbon dioxide and produce biomass essential for life on earth. Photosystem integrity and activity are negatively impacted by fluctuations in incident light from the sun. How plants regulate photosynthetic dynamics under natural fluctuating growth light is relatively poorly understood. Loss of the Arabidopsis thaliana chloroplast lumenal protein MPH2 causes photosystem II (PSII) repair deficiency under changing light. PSII repair mutants are impaired in growth under greenhouse fluctuating light environments, while photoprotection mutants grow normally. These findings inform strategies for engineering plant photosynthetic performance under field conditions, to sustainably address increasing needs for food, fiber, and fuel at a time of changing climate and rapid population growth. Author contributions: J.L. and R.L.L. designed research; J.L. performed research; J.L. and R.L.L. analyzed data; and J.L. and R.L.L. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Freely available online through the PNAS open access option. Data deposition: The homozygous mutant lines of mph2-1 and mph2-2 reported in this paper have been deposited in the Arabidopsis Biological Resource Center (ABRC) (accession nos. CS69598 and CS69599, respectively). 1

To whom correspondence should be addressed. Email: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1712206114/-/DCSupplemental.

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Edited by Winslow R. Briggs, Carnegie Institution for Science, Stanford, CA, and approved August 4, 2017 (received for review July 10, 2017)

that regulates PSII repair to maintain efficient photosynthesis and normal growth. Analysis of mph2 and previously described PSII repair- and photoprotection-defective mutants grown in the greenhouse revealed that repair—but not protection—is required for normal vegetative growth under dynamic environmental conditions. Results Identification of a Mutant with Defects in Photosynthetic Regulation Under Light Stress Conditions. Study of photoautotrophic organ-

isms under well-controlled growth light conditions led to the discovery and characterization of a variety of photosynthetic regulatory proteins (13–16). Most of these proteins are located in the chloroplast stroma or thylakoid membrane, and relatively few are in the lumen; in fact, the functions of the majority of lumen-targeted proteins are unknown (15, 23). To seek lumenal proteins with roles in photosynthetic regulation, we screened for A. thaliana mutants of thylakoid lumenal proteins with growth and photosynthetic defects under changing light conditions. We sought proteins of unknown function that are coregulated with previously established regulatory factors by exploring functional protein association networks in the STRING database (23, 24) (SI Appendix, Fig. S1). We focused on genes of unknown function that encode proteins annotated to be in the lumen based upon Arabidopsis proteomics data (Plant Proteome Database, ppdb.tc.cornell.edu) (25). Transfer DNA (T-DNA) insertion mutants of the candidate genes grown under standard growth chamber light conditions (100 μmol·m−2·s−1, referred to as “growth light” throughout) were shifted to high light (1,000 μmol·m−2 ·s −1) for 3 h and analyzed for maximum photochemical efficiency of PSII (Fv/Fm). Complete loss-of-function mutants (SI Appendix, Fig. S2) in the gene At4g02530/MPH2 (hereafter named mph2-1 and mph2-2) had significantly lower Fv/Fm, Fm, and oxygen evolution activity and higher Fo compared with the wild-type parent under high-irradiance light (Fig. 1 and SI Appendix, Table S1). In contrast, PSI activity, which is measured by far-red oxidizable P700, was quite similar between mph2 mutants and the wild type

Fig. 1. Loss of MPH2 protein renders PSII sensitive to increased light fluence. (A) Growth phenotypes of mph2-1 mutant and the corresponding Col-0 wild type as well as mph 2–2 and Ws-4 wild type grown under standard growth light (GL, 100 μmol·m−2·s−1) conditions. (B–D) False-color images and numerical values representing Fv/Fm (B) under growth light, (C) after a 3-h high-light (HL, 1,000 μmol·m−2·s−1) treatment, and (D) after a 2-d high-light treatment. Red pixels in C and D indicate that Fv/Fm is below the cutoff value (0.58 and 0.68, respectively). Wild type and mutant t test comparison statistics are shown (means ± SD; n = 3). **P < 0.01; ns, no significant difference from the wild type.

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under growth light or high light (SI Appendix, Table S1). These results demonstrate that loss of MPH2 gene function causes reduced PSII efficiency following a shift from growth light to photoinhibitory light conditions. To test whether mph2 mutants are also susceptible to longer term high-light stress, plants were shifted from growth light and maintained under high light for 2 d (SI Appendix, Fig. S3). As seen for short-term high-light treatment, both mutants displayed significantly decreased Fv/Fm compared with the wild type (Fig. 1D). These results are consistent with STRING network analyses, where MPH2 was associated with the lumenal photosynthetic regulatory factors CYP38, PPL1, TLP18.3, and ROC4/CYP20-3 as well as the thylakoid membrane factors LQY1 and HHL1 (SI Appendix, Fig. S1). Loss-of-function mutants in these genes all exhibit reduced PSII efficiency upon exposure to short-term or long-term high-light treatments (26–34). BLAST searches with full-length inferred MPH2 protein sequences revealed that MPH2 is evolutionarily conserved among oxygenic photosynthetic eukaryotes; all have predicted N-terminal chloroplast transit peptides followed by lumenal transit peptides (SI Appendix, Fig. S4A). Subcellular fractionation of chloroplast compartments validated that Arabidopsis MPH2 is located in the chloroplast thylakoid lumen (SI Appendix, Fig. S4 B–D), strengthening published proteomic analyses (25, 35, 36). Collectively, these data are consistent with the hypothesis that MPH2 is a green lineage-specific thylakoid lumen protein required for photosynthetic acclimation of PSII to stressful light conditions. MPH2 Is Required for Normal Growth Under Fluctuating Light Conditions. The observation that mph2 mutant PSII is susceptible

to shifts from growth to high light and the fact that plants grow under environments of short- and long-term light fluctuations prompted us to ask if MPH2 has a role in regulating photosynthesis under fluctuating light intensity. We took several approaches to probe the function of MPH2 in fluctuating light environments. First, dark-adapted plants were illuminated with alternating cycles of low light (55 μmol·m−2·s−1) and high light (700 μmol·m−2·s−1)— this revealed increasingly reduced effective quantum yield of PSII [Y(II)] in mph2 mutants compared with the wild type after each light transition (Fig. 2A). These results suggest that the preceding light environment may influence the function of MPH2. This led us to test whether MPH2 has a role in dark-to-light transitions. Darkadapted plants were exposed to low light, growth light, or high light, respectively, and then returned to the dark. Surprisingly, mph2 mutants exhibited wild type-like PSII efficiency under all conditions tested (SI Appendix, Fig. S5). These results suggest that MPH2 is required for photosynthetic acclimation to continuously fluctuating light. We next examined the physiological importance of MPH2 under fluctuating light environments. Growth light-grown wildtype and mutant plants were moved to the greenhouse where they experienced dynamic changes in light intensity and temperature. As shown in Fig. 2B, greenhouse-grown mph2 mutants were smaller than the wild type. Similar phenotypes were observed for mph2 in different seasons of the year despite differences in day length, cloud cover, and temperature (SI Appendix, Fig. S6). Moreover, the observed mph2 growth retardation correlated with significant reduction of PSII efficiency and oxygen evolution activity (Fig. 2C and SI Appendix, Table S1), consistent with the reduced Y(II), ETR(II), Fm′/Fm, and F′/Fm seen under artificial fluctuating light (Fig. 2A and SI Appendix, Fig. S7). In contrast, photooxidizable P700 was the same in greenhousegrown mutant and wild-type plants (SI Appendix, Table S1). Together, these results are consistent with the hypothesis that MPH2 contributes to maintaining photosynthetic efficiency of plants under fluctuating light conditions. Liu and Last

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role in regulating PSII repair under changes in light conditions (Fig. 3D).

Fig. 2. Susceptibility of mph2 mutant plants to continuously fluctuating light and greenhouse fluctuating light environments. (A) Photochemical quantum yield of PSII as a measurement of photosynthetic electron transport was decreased in mph2 mutants upon exposure to continuously fluctuating light. Two hundred and forty seconds of low light (LL, 55 μmol·m−2·s−1) and 120 s of high light (HL, 700 μmol·m−2·s−1) alternately cycled, mimicking fluctuating growth light conditions. (B) Representative images showing stunted growth of mph2 mutant plants following a shift of 17-d-old seedlings grown under constant light intensity (100 μmol·m−2·s−1) in growth chambers to greenhouse fluctuating light conditions on October 16, 2015, followed by 18 d of greenhouse growth. (C) The retarded growth of mph2 mutant plants associated with reduced PSII efficiency. False-color images representing Fv/Fm of B. Red pixels indicate that Fv/Fm is below the cutoff value of 0.67. Wild type and mutant t test comparison statistics are shown (means ± SD; n = 6). **P < 0.01.

Lack of MPH2 Impairs PSII Repair. To gain insight into how defects in photosynthetic energy conversion caused the reduced growth of mph2 mutants under greenhouse changing light conditions, we asked whether plants suffered from photodamage to PSII or repair deficiency upon loss of MPH2. The approach taken to test this hypothesis was to monitor the extent of photodamage and capacity for PSII repair. We conducted in vitro photoinhibition assays in which the repair of PSII was blocked by the inhibitor of plastid protein synthesis lincomycin. In the absence of lincomycin, mph2 mutants exhibited a more severe reduction in PSII activity than the wild type during high-light illumination (Fig. 3A), which is consistent with the results of in planta 3-h or 2-d high-light treatments (Fig. 1 C and D). In contrast, no difference between mph2 and wild type was seen in the presence of lincomycin (Fig. 3B). This suggests that MPH2 is required for the repair of PSII when subjected to light stress. To further explore the hypothesis that MPH2 is required for PSII repair, we analyzed the recovery of the mph2 mutants from high-light stress-induced damage. As observed for the PSII repair-defective lqy1-1 mutant, mph2 mutants recovered more slowly and Fv/Fm did not return to the original level following shift to growth light for 5 h. In contrast, the PSII photoprotectiondefective npq4 mutant displayed a higher recovery rate (8, 32) (Fig. 3C). To ask whether the delayed recovery observed in the mutants was caused by excessive PSII photoinactivation following high-light treatment, the mutant and the wild type were treated with enough high-irradiance illumination to induce similar amounts of photoinhibition and allowed to recover under growth light. The observation that PSII activity recovered more slowly in the mutants is further evidence that MPH2 plays a Liu and Last

repair deficiency in mph2 mutants, we examined the oligomeric state of photosynthetic complexes using blue-native PAGE (BNPAGE). The intensities of the bands corresponding to PSII supercomplexes and PSI/PSII dimer were decreased in mph2 mutants subjected to high-irradiance light compared with the wild type (SI Appendix, Fig. S8A). We next performed immunoblot analysis of 2D BN/SDS PAGE gels to examine the distribution and abundance of individual protein subunits in various PSII complexes. These results revealed marked increases in the accumulation of protein subunits within PSII monomers (“PSII-M” and “RC-47”) in samples from mutant plants subjected either to high-irradiance light or to greenhouse fluctuating light (Fig. 4A and SI Appendix, Fig. S9B and Table S2). This was accompanied by concomitant strong reductions in the accumulation of protein subunits within PSII supercomplexes. In addition, the free CP43 detected in the wild type was barely seen in mph2 mutants (Fig. 4A). However, the overall steady-state levels of PSII proteins were very similar between mph2 and wild type (SI Appendix, Figs. S8 B and C and S10B), despite the observation that the mutant had significant reduction of PSII activity under high-light stress as well as in the greenhouse (Figs. 1 C and D and 2C and SI Appendix, Table S1). These data led us to examine whether the turnover of PSII proteins is affected in the mutants. Detached leaves of plants infiltrated with or without lincomycin were subjected to highillumination light, and the amounts of PSII proteins were analyzed. As expected, both D1 and D2 reaction center proteins were dramatically reduced in the wild type (Fig. 4B). In contrast, steady-state levels of D1 and D2 were only mildly reduced in the mph2 mutants despite lack of synthesis of these proteins. These

Fig. 3. PSII repair is deficient in mph2 mutants. (A) In vitro photoinhibition assay showing differences in PSII activity in mph2 mutants and their wildtype counterparts. Detached leaves of the wild-type and mph2 mutant plants were illuminated with high-irradiance light (1,000 μmol·m−2·s−1) for indicated times. (B) mph2 mutants displayed comparable sensitivity to that of the isogenic wild type to photoinhibitory light in the presence of chloroplast translation inhibitor lincomycin. (C) Time course of recovery of Fv/Fm following PSII photoinhibition. The wild-type, mph2, lqy1-1, and npq4 mutant plants were shifted from growth light to high light (HL, 1,000 μmol·m−2·s−1) for 3 h and subsequently transferred back to growth light to allow PSII activity recovery. (D) Delayed recovery of PSII activity in mph2 mutants. The wild type and the mutant were high light-treated to 50% of growth-light PSII activity and subsequently shifted to growth light to allow recovery.

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MPH2 Promotes PSII Protein Subunit Turnover and Interacts with Core Complexes During PSII Repair. To identify the cause(s) of PSII

complexes is consistent with the hypothesis that loss of MPH2 disrupts PSII monomer disassembly during PSII repair. Two approaches were taken to test the hypothesis that MPH2 plays a role in PSII monomer disassembly. First, the association of MPH2 protein with PSII was analyzed by immunoblot analysis of 2D BN/ SDS PAGE gels. The results showed that a subset of MPH2 protein was found in the region of the gel corresponding to monomeric PSII complexes under high light (Fig. 4A) and the bulk of it comigrated with monomers under fluctuating light (SI Appendix, Fig. S9B). This indicates that MPH2 protein copurifies with PSII monomers. Next we examined the interaction of MPH2 protein with PSII by coimmunoprecipitation assays. As shown in Fig. 4C, D1, D2, CP43, and CP47 subunits of PSII coprecipitated using anti-MPH2 antibody, while ATPase subunit CF1β, Cytochrome b6f Cyt. f protein, and PSI PsaA did not. The specificity of the interaction of MPH2 with PSII core complexes was reinforced by the observation that MPH2 did not coprecipitate with MPH1 (Fig. 4C), which is a PSII core protein-associated auxiliary factor that protects PSII against photodamage (37, 38). These coprecipitation results support the hypothesis that MPH2 is involved in PSII repair. Deficiency in PSII Repair, but Not Photoprotection, Correlates with Reduced Growth Under Greenhouse Light Environments. The find-

Fig. 4. Analysis of PSII complexes, subunit stability, and interactions between MPH2 and photosynthetic proteins. (A) Overaccumulation of PSII monomers in mph2 mutants as revealed by immunoblot analysis of 2D BN/SDS/ PAGE. Chlorophyll–protein complexes were prepared from leaves of high lighttreated wild-type and mph2 mutant plants, fractionated by 2D BN/SDS/PAGE, and detected by specific antibodies as indicated to the right. (B) Lincomycin treatment causes differences in PSII reaction center protein stability in mph2 mutants compared with wild type. Relative protein abundance of each thylakoid sample is given below, and the level of proteins from each genotype without adding lincomycin is designated as 100%. Shown below is Coomassie Brilliant Blue (C.B.B.) staining of a reference SDS/PAGE gel. (C) Coimmunoprecipitation analysis demonstrating interactions between MPH2 and PSII core protein complexes. Anti-MPH2 antiserum coupled to protein A/G agarose was incubated with thylakoid samples, and the immunoprecipitated proteins were probed with antibodies as indicated to the right.

results indicate that the degradation of PSII proteins in mph2 mutants is impaired during PSII repair. To ask whether the reduced degradation rate of PSII reaction center proteins seen in the mutant is correlated with changes in thylakoid-localized proteases, we analyzed the abundance of FtSH and Deg8. As shown in SI Appendix, Fig. S10A, no changes were detected in the mph2 mutants either under controlled photoinhibitory light or fluctuating light conditions. The syndrome of reduced mph2 mutant PSII reaction center protein turnover combined with accumulation of PSII monomeric 4 of 8 | www.pnas.org/cgi/doi/10.1073/pnas.1712206114

ings that loss of MPH2 perturbs PSII repair and causes reduced greenhouse plant growth suggest that MPH2-mediated PSII repair plays a role in regulating plant adaptation to dynamic light environments. This led us to ask whether the reduced growth and PSII efficiency under greenhouse growth conditions is a general property of light-sensitive PSII-defective mutants. Mutants previously demonstrated to be deficient in PSII repair—lqy1-1, tlp18.3, and psb27—along with the PSII photoprotection-defective mph1-1 mutant (29, 32, 33, 37–40) were grown in the greenhouse to compare their responses to dynamic growth light conditions. We found that all PSII repair mutants exhibited decreased greenhouse growth, albeit to different extents (Fig. 5A and SI Appendix, Fig. S11). In contrast, the PSII protection-defective mph1-1 mutant showed wild type-like growth, despite a significant reduction in PSII activity. As previously reported (29, 32, 33, 37– 40), none of these mutants had growth defects under standard growth light conditions (SI Appendix, Fig. S12 A and B). It is noteworthy that tlp18.3 did not have significantly lower Fv/Fm than wild type (Fig. 5B). The observation that the growth of mph1-1 was normal under fluctuating environmental conditions led us to examine the responses of other PSII photoprotection-defective mutants to greenhouse growth conditions. The npq1 and npq4 mutants were selected because they have severe defects in photoprotective energy dissipation and also showed wild type-like growth under standard growth light conditions (8, 41) (SI Appendix, Fig. S12 C and D). As seen with mph1-1, neither npq1 nor npq4 mutants displayed an appreciable growth decrease in the greenhouse, despite a significant reduction in Fv/Fm (Fig. 5 C and D and SI Appendix, Fig. S13). To further test the importance of PSII repair processes under fluctuating light, we analyzed the three additional PSII repairdefective mutants met1-2, cyp20-3/roc4, and deg8, which all had wild type-like growth and PSII efficiency under growth light (SI Appendix, Fig. S14 A and B), as previously reported (30, 31, 42, 43). In contrast, these mutants displayed reduced growth and PSII activity in the greenhouse (SI Appendix, Fig. S14 C–F). Taken together, these data lead us to propose that PSII repair-defective mutants—but not photoprotection-defective mutants—are generally impaired in growth acclimation under dynamic environments. Discussion Photosynthesis plays a crucial role in biology and biogeochemistry, transforming sunlight energy and CO2 into the metabolic products Liu and Last

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of life. However, incident solar irradiance varies dramatically, changing seasonally, diurnally, and with rapid shading by cloud cover or canopy leaf movement. Photosynthetic physiology shows remarkable resilience to these unpredictable light fluctuations, raising strong interest in the biochemical mechanisms behind these adaptations. Here we document the role of the Arabidopsis nuclearencoded chloroplast-targeted thylakoid lumenal protein MPH2 in maintaining photosynthetic performance both under changing light conditions and static high-irradiance light. Our experiments reveal that MPH2 is a required component of the PSII repair machinery—possibly involved in disassembling monomeric complexes—and that PSII repair correlates with acclimation of plants to natural environments. Network Analysis Identified MPH2 as a Thylakoid Lumenal PSII Repair Candidate. Thus far, proteomic analyses identified more than

80 lumenal thylakoid proteins in Arabidopsis (35, 36), with functions described for very few (23, 44). We used a network approach to find lumenal proteins of unknown function coregulated with previously characterized lumenal photosynthetic regulatory factors (23, 24) and identified MPH2 as matching the search criteria. Our biochemical and physiological results lead us to propose that the thylakoid lumenal MPH2 protein plays a role in PSII repair induced by a shift to photoinhibitory light conditions. The identification of a lumen-targeted factor involved in regulating photosynthesis suggests that the combined network approach and mutant analysis can continue to extend our understanding of photosynthetic regulation in plants and microbes. MPH2 Protein Is Required for PSII Repair Under Changing Light Conditions. Our results argue that MPH2 protein is required for

PSII repair to maintain proper photosynthetic function under changing light conditions. Growth of mph2 null mutants was indistinguishable from wild-type plants under well-controlled “normal growth light” (Fig. 1A and SI Appendix, Fig. S3). This is consistent with the very similar PSII efficiency, photosynthetic protein abundance, and functional complex assembly in the mutant compared with wild type (Fig. 1B and SI Appendix, Figs. S8 A and B and S9A). In contrast, mph2 mutant PSII efficiency was reduced relative to wild type under short- and long-term high-light stress and also in greenhouse fluctuating light (Figs. Liu and Last

1 C and D and 2C and SI Appendix, Table S1). Moreover, mph2 mutants had delayed recovery of PSII activity following a shift from high-irradiance to growth light (Fig. 3 C and D), arguing that mph2 mutants are defective in PSII repair. Consistent with this hypothesis, MPH2 protein was found mainly in the stromal lamella fraction (SI Appendix, Fig. S4D), where repair of damaged PSII complexes takes place (14–16). Direct evidence for defective repair under photoinhibitory light came from in vitro photoinhibition assays with the chloroplast protein synthesis inhibitor lincomycin (Fig. 3 A and B). Further biochemical assays with BN-PAGE and 2D BN/SDS PAGE immunoblot analysis revealed that mph2 mutants are impaired in formation of PSII supercomplexes during PSII repair (Fig. 4A and SI Appendix, Figs. S8A and S9B and Table S2). Evidence That MPH2 Protein Is a PSII Repair Disassembly Factor. Although a variety of PSII repair/assembly factors have been characterized (13–16), to the best of our knowledge no other proteins are described as being involved in disassembling PSII monomers during repair. Our data are consistent with the hypothesis that the mph2 mutant PSII deficiency results from impaired turnover of PSII protein subunits caused by a defect in disassembly and repair of damaged monomeric complexes. Multiple lines of evidence are consistent with this model. First, unlike the PSII repair-defective mutants lqy1, hhl1, and met1— where total amounts of PSII core proteins decrease under highirradiance light (32–34, 42)—mph2 susceptibility to photoinhibitory light was not associated with overall reduction in PSII reaction center proteins (SI Appendix, Figs. S8 B and C and S10B). Second, 2D BN/SDS PAGE immunoblot analysis revealed that monomeric PSII complexes overaccumulated in mph2 mutants subjected either to high-light treatment or fluctuating light while PSII supercomplexes decreased (Fig. 4A and SI Appendix, Fig. S9B and Table S2). Furthermore, the free CP43 observed in the wild type was barely detectable in the mutant (Fig. 4A). These data are consistent with two contrasting hypotheses: that loss of MPH2 protein perturbs the disassembly of lower order monomeric PSII or inhibits reassembly of monomers to dimeric PSII complexes. To discriminate between these alternatives, we examined the stability of PSII complexes by assessing turnover rates of their PNAS Early Edition | 5 of 8

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Fig. 5. Mutants deficient in PSII repair have impaired growth under greenhouse conditions. (A and C) Representative images showing reduced growth of PSII repair-defective mutants mph2, lqy1-1, tl18.3, and psb27 in the greenhouse, with photoprotection-defective mutants mph1-1, npq1, and npq4 exhibiting no abnormal growth phenotypes. Seventeen-day-old seedlings grown under constant light intensity in growth chambers were transferred to greenhouse fluctuating light conditions on June 17, 2016 for 11 d (A) and April 20, 2016 for 12 d (C), respectively. (B and D) Analysis of PSII activity of greenhouse-grown wild-type and mutant plants, and false-color images representing Fv/Fm of A and C, respectively. Red pixels in D indicate that Fv/Fm is below the cutoff value of 0.68. Wild type and mutant t test comparison statistics are shown (means ± SD; n = 4). **P < 0.01; ***P < 0.001; ns, no significant difference from Col-0 wild type. The psb27 used here is the SALK 004769 line, which contains a second knockout mutation in CP26 (40).

component proteins under high-irradiance light. The observation that D1 and D2 PSII reaction center proteins stably accumulated in mph2 mutants—both in the presence and absence of chloroplast de novo protein synthesis—provides direct evidence that degradation of PSII core proteins was impaired (Fig. 4B). In contrast, loss of the PSII supercomplex reassembly factors LQY1, HHL1, or MET1 caused accelerated rates of D1 and D2 degradation under high-light stress (32–34, 42). Because PSII monomer disassembly precedes damaged reaction center degradation and replacement (13–16), the failure of mph2 mutants to turn over PSII reaction center components under stressful light implicates MPH2 in monomer disassembly. Moreover, the decreased degradation rates of D1 and D2 did not result from the lack of FtSH and Deg8 proteases involved in damaged PSII protein degradation (SI Appendix, Fig. S10A). This, in turn, is consistent with the hypothesis that the observed mph2 degradation defect is due to impairment in PSII monomer disassembly, which is required for subsequent repair and reassembly. Finally, MPH2 protein copurifies with PSII monomers and interacts directly with PSII core complexes, as expected for a protein playing a role in monomeric PSII complex disassembly (Fig. 4 A and C and SI Appendix, Fig. S9B). Taken together, these data lead us to propose that MPH2 protein is a thylakoid lumen PSII monomer disassembly factor. PSII Repair Mutants Are Defective in Growth Acclimation Under Dynamic Light Conditions. The findings that mph2 mutants showed

lower Y(II) than the wild type under artificial fluctuating light and exhibited reduced growth in the greenhouse led us to explore regulatory mechanisms ensuring optimal plant performance under natural light conditions. We used greenhouse growth conditions because these subject plants to rapid, daily, and longer term variations in light, temperature, and humidity, approximating a natural setting. As similar results were obtained in studies replicated over multiple seasons and years, we propose that dynamic changes in light irradiance are responsible for the observed impaired greenhouse growth of PSII repair-defective mutants mph2, lqy1-1, tlp18.3, psb27, met1-2, cyp20-3/roc4, and deg8 (Figs. 2 and 5 and SI Appendix, Figs. S6, S11, and S14). Our data are consistent with published reports that—under growth chamber fluctuating light conditions—tlp18.3, psb27, and met1-2 displayed stunted growth phenotypes (29, 40, 42, 45). Moreover, reduced greenhouse growth is not simply the result of lower PSII efficiency. First, PSII photoprotection-defective mutants mph1, npq1, and npq4 had no appreciable decrease in greenhouse growth compared with wild type, despite lower PSII activity (Fig. 5 and SI Appendix, Figs. S13 and S14 C–F). These data support published results demonstrating normal growth of npq1 and npq4 mutants under growth chamber fluctuating light conditions (46–48). In contrast, the PSII repair-defective mutants were smaller than wild type in the greenhouse, and all but one had significant reductions in PSII activity (Figs. 2 and 5 and SI Appendix, Figs. S6, S11, and S14). The exceptional mutant—tlp18.3—had normal Fv/Fm in our greenhouse studies (Fig. 5B and SI Appendix, Fig. S14F), corroborating published growth chamber fluctuating light results (45). TLP18.3 is unusual compared with the other repair factors, having been proposed to have a role in PSII repair-mediated defense response rather than repair per se (45). Collectively, these data suggest that PSII repair is a regulatory mechanism that confers adaptive advantages to plants under dynamic light environments. Repair Versus Protection of PSII Under Fluctuating Light Environments.

Plant photosynthesis adapts to enormous light fluctuations in native habitats and agricultural ecosystems, with multiple short- and long-term regulatory mechanisms influencing the light acclimation capacity of the photosynthetic machinery. This multitiered regulatory scheme for fluctuating light adaptation is not surprising given the importance and complexity of the two photosystems. 6 of 8 | www.pnas.org/cgi/doi/10.1073/pnas.1712206114

This regulatory complexity is illustrated when considering studies with NPQ-deficient npq1 and npq4 mutants. The observation that npq plants showed normal growth under greenhouse and growth chamber fluctuating light conditions (46–48) (Fig. 5C and SI Appendix, Figs. S13 and S14) suggests that NPQ deficiency does not have a deleterious effect on plant vegetative growth acclimation under long-term fluctuating light conditions. In contrast, NPQ was demonstrated to play a crucial role in rapid adjustments of photosynthesis to artificial fluctuating light and shading (49–52). Although npq mutants suffered higher photoinhibition and had less seeds relative to the wild type in field experiments, they did not display visible vegetative growth defects (53, 54). The state transition mutant stn7 behaved similarly under these same field conditions, with lower seed yield and normal vegetative growth (54). Analysis of the stn7 mutant revealed PSI as a target of damage under rapidly fluctuating white light: stn7 plants exhibited PSI damage associated with reduced growth (47). In addition, severe defects in PSI and growth were seen in the pgr5 mutant under artificial fluctuating light conditions and in the field (11). The PGR5 protein was proposed to safeguard PSI by preventing uncontrolled bursts of reducing equivalents from PSII (11, 55). The results presented in this work are consistent with the hypothesis that PSII is the direct target of damage in the absence of repair factor MPH2. The accumulation of PsaA protein as well as PSI activity was not affected in mph2 mutants under either controlled photoinhibitory light or greenhouse fluctuating light (SI Appendix, Figs. S8 B and C, S9B, and S10 B and C and Table S1). Moreover, all PSII repair-defective mutants that exhibited stunted greenhouse growth had a wild-type level of PsaA protein (SI Appendix, Fig. S10C). In contrast, the values of Fv/Fm, Fm, Y(II), ETR(II), Fm′/Fm, and F′/Fm as well as oxygen evolution activity were all lower and Fo was higher in mph2 mutants (Figs. 1 and 2 and SI Appendix, Fig. S7 and Table S1). This also is consistent with the result that MPH2 interacts with PSII but not with the other photosynthetic complexes (Fig. 4C). These data, combined with extensive documentation of the importance of PSII protection and repair, argue that under natural dynamic environmental conditions photosynthetic efficiency and growth enhancements by breeding or engineering will require concerted manipulation of multiple light acclimation mechanisms while maintaining balanced electron transport between PSII and PSI (Fig. 6).

Fig. 6. A simplified model depicting the correlation between regulation of photosynthetic light reaction and plant growth acclimation under fluctuating light environments. (A) Fluctuating light causes excessive photodamage in PSI due to lack of photoprotection, thus lowering photosynthetic efficiency and leading to reduced vegetative growth. (B) Fluctuating light affects normal PSII function due to defects in repair, thus lowering overall photosynthetic electron transport rate and leading to reduced vegetative growth. (C) Operation of multiple light acclimation mechanisms, such as PSII repair and PGR5-mediated PSI protection, ensures normal photosynthetic capacity and normal growth under fluctuating light environments. (D) On the basis of this study and the work of others, we hypothesize that plant fitness in the field conditions would be enhanced by properly manipulating light acclimation mechanisms to increase efficiencies of both PSII and PSI while ensuring a balanced photosynthetic electron flow. Blue and pink colors denote the proportion of active and inactive photosystems, respectively.

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Materials and Methods Plant Materials and Growth Conditions. A. thaliana ecotype Columbia background T-DNA insertion line for At4g02530, mph2-1 (SAIL_750_E07) was obtained from the Arabidopsis Biological Resource Center (ABRC; https://www. arabidopsis.org/), and ecotype Wassilewskija (Ws) background T-DNA insertion line for At4g02530, mph2-2 (FLAG_567F06) was from the Versailles Arabidopsis Stock Center (INRA; publiclines.versailles.inra.fr/). Homozygous mutants were identified by genomic PCR with specific primers as listed in SI Appendix, Table S3. The homozygous mutant lines of mph2-1 and mph2-2 used in this study were deposited in the ABRC under accession nos. CS69598 and CS69599, respectively. Columbia background T-DNA insertion lines for At5g07020 (mph1-1, SALK_143436), At1g75690 (lqy1-1, SALK_021824), At1g54780 (tlp18.3, SALK_ 109618), At1g03600 (psb27, SALK_004769), At1g55480 (met1-2, WISCDSLOXHS212_ 08F), At3g62030 (cyp20-3/roc4, SALK_001615), and At5g39830 (deg8, SALK_ 004770), and deletion lines for At1g08550 (npq1) and At1g44575 (npq4), were identified in the Chloroplast 2010 Project (https://plastid.natsci.msu.edu/) (32, 56, 57). The SALK 004769 line contains mutations in two genes (Fig. 5 and SI Appendix, Figs. S11 and S12)—a T-DNA insertion in At1g03600 and a point mutation in CP26 (At4g10340)—and each mutation causes a complete loss of PSB27 or CP26 protein, respectively (40). The single knockout psb27 and cp26 mutants were kindly provided by Sheng Luan, University of California, Berkeley, and were used in experiments shown in SI Appendix, Fig. S14. The mph1-1, lqy1-1, tlp18.3, cp26, met1-2, cyp20-3/roc4, and deg8 knockouts were as previously described (29–33, 37–40, 42, 43). The npq1 and npq4 knockouts were identical to npq1-2 and npq4-1, respectively, which have been previously reported (8, 41, 46, 47). Seeds were sown on soil (LC1 Sunshine mix; Sun Gro Horticulture) and stratified in the dark at 4 °C for 3 d and then grown in standard controlled growth chambers as previously described, except that the photoperiod was a 10-h light/14-h dark cycle (32, 38). To evaluate the effects of variation in light irradiance on growth and photosynthetic performance of Arabidopsis wild-type and mutant plants, multiple independent greenhouse experiments were conducted in 3 consecutive years (2015, 2016, and 2017) at Michigan State University, East Lansing (42.72°N, 88.21°W). Light intensity varied with incoming irradiance during the daytime. Seventeen-day-old seedlings grown in standard controlled growth chambers were moved to the greenhouse. Sixteen plants per genotype were used in each greenhouse experiment. Pots (one plant per pot) were arranged in a randomized design (generated by Microsoft Excel 2011) in trays placed on greenhouse tables, with the trays rotated every other day.

In Vitro Photoinhibition Assays and Light Stress Recovery Treatments. For photoinhibitory analyses, detached leaves of the wild-type and mutant plants were infiltrated with 1 mM lincomycin (Sigma-Aldrich) solution or water (38) and then illuminated under an intensity of light of 1,000 μmol·m−2·s−1 at indicated times. The temperature was maintained at 21 °C during the highlight treatment. For the light stress recovery treatments, whole plants were exposed to an irradiance light of 1,000 μmol·m−2·s−1 for 3 h, or detached leaves were illuminated with a light intensity of 1,200 μmol·m−2·s−1, and Fv/Fm was monitored during the high-light treatment so that the Fv/Fm values of both the wild type and the mutant were reduced to ∼50% of the original levels and subsequently shifted to growth light (100 μmol·m−2·s−1) to analyze photoinhibition recovery. Protein Analyses. Immunoblotting was performed as previously described (38). Thylakoid membrane proteins of Arabidopsis rosette leaves with equivalent chlorophyll were resolved by 12.5% SDS/PAGE and transferred to nitrocellulose membranes (GE Healthcare). Primary antibodies raised against photosynthetic proteins were purchased from Agrisera. Blots were detected using Clarity Western ECL Substrate and visualized by Image Lab (Bio-Rad). Affinity-purified anti-MPH2 polyclonal antibodies were produced by Thermo Fisher Scientific. A 20-amino acid peptide (corresponding to amino acids KNDIESSKLAFVSSAGAFEK) of MPH2 protein with an additional N terminus Cys residue was synthesized, conjugated with keyhole limpet hemocyanin, and used to generate rabbit MPH2 antibody. For BN-PAGE analysis, preparation and solubilization of intact thylakoids were performed as previously described (44). Electrophoresis was carried out by using Native PAGE Novex4–16% Bis–Tris mini gel and XCellSureLock minicell at 4 °C (Thermo Fisher Scientific). 2D BN/SDS/PAGE was performed as previously described (38). Analyses of Protein Degradation. For analysis of PSII protein stability, detached leaves of the wild-type and mutant plants infiltrated with 1 mM lincomycin (Sigma-Aldrich) solution or water were exposed to high-irradiance light (1,200 μmol·m−2·s−1) for 2 h (38). Thylakoid membrane proteins extracted from the detached leaves were used for immunoblot analysis. The temperature was maintained at 21 °C during the high-light treatment. Subcellular Localization of MPH2 and Coimmunoprecipitation Assays. Immunolocalization and trypsin treatment of intact thylakoids were carried out as previously described (44). Subfractionation of thylakoids into grana margin, grana core, and stroma lamellae was performed as described in ref. 32. Coimmunoprecipitation assays using anti-MPH2 antiserum were performed as described in ref. 38.

Photosynthetic Parameter Measurements. Chlorophyll fluorescence parameters were measured with the MAXI version of the IMAGING-PAM M-Series chlorophyll fluorescence system (Heinz-Walz Instruments) as described previously (56). Plants were dark-adapted for 30 min, and minimum fluorescence (Fo) and maximal fluorescence (Fm) in the dark-adapted state were measured. The maximal photochemical efficiency of PSII was determined as Fv/Fm = (Fm − Fo)/Fm. For the measurements of photochemical quantum yield of PSII and ETR(II) under controlled fluctuating light, actinic light was applied on leaves of whole plants grown under growth light. Two hundred and forty seconds of low light

ACKNOWLEDGMENTS. We thank members of the R.L.L. laboratory and Yan Lu and her research group at Western Michigan University for valuable suggestions and Shin-Han Shiu (Michigan State University) for fruitful discussions. We are grateful to Dr. Jiying Li (Jianping Hu lab, Michigan State University) for access to the high-light chamber, to David A. Hall and Geoffry A. Davis (David M. Kramer lab, Michigan State University) for assistance in acquiring photosynthesis data, and to Prof. Sheng Luan (University of California, Berkeley) for providing psb27 and cp26 mutant seeds. We acknowledge the ABRC and the INRA for providing seed stocks. This work was supported by National Sciences Foundation Grants Molecular and Cellular Biosciences (MCB)-1244008 and MCB1119778.

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(55 μmol·m−2·s−1) was interrupted by 120 s of high light (700 μmol·m−2·s−1). F′/Fm is an indicator of the relative QA redox state in non-steady state conditions (11), where F′ is the fluorescence yield under actinic light. Oxygen evolution measurements using isolated thylakoid membranes were performed as described by Ishihara et al. (28). The light-induced in vivo absorbance changes of P700 at 820 nm were measured as described by Liu et al. (44).

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Concluding Remarks This study extends our understanding of the elaborate PSII repair system in higher plants and reveals that repair is not only required for PSII maintenance under constant high-light conditions but is also a regulatory mechanism facilitating plant adaptation to fluctuating sunlight. Given that PSII repair is common to all oxygenic photosynthetic organisms, testing its importance to algae and cyanobacteria under fluctuating light conditions will add insight into the evolution of photosynthesis. These findings implicate that informed manipulation of PSII repair can be a strategy for improving photosynthetic productivity and plant performance in natural environments to sustainably meet the growing demands for food, fuel, and fiber.

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